Purpose: :
Sigma receptors have no known homology with other receptorsystems, have no known natural ligands, but appear to play acritical role in a large diversity of cell functions. In theabsence of a conventional pharmacology, siRNA technology providesa direct means of elucidating the major cell signalling pathwaysinfluenced by this receptor system.

Methods: :
The non–transformed human lens cell line FHL124expresses the sigma–1 receptor (Sig–1R) and wasemployed for these studies. FHL124 cells were maintained inserum–free Eagle’s minimum essential medium (EMEM)or EMEM supplemented with 5% fetal calf serum (FCS) at 350Cin 5% CO2. Two independent Sig–1R siRNAs and universalscrambled control siRNA were designed and generated by Ambionand cells were transfected in standard Oligofectamine medium.Cell growth was assessed by measuring total protein and celldeath by quantifying LDH leakage. Expression of the Sig–1Rwas assayed by RT–PCR. Western immunoblot techniques wereemployed to measure levels of Sig–1R protein, pERK/ERK,pAkt/Akt and caspase–3.

Results: :
72 hours of transfection with either of the two siRNAsdirected against Sig–1R reduced messenger RNA and proteinlevels by over 70 and 60% respectively. Subsequent incubationfor 96 hours in culture medium (EMEM) supplemented with 5% serumgave a partial recovery of message, but there was no significantincrease in protein expression. LDH leakage assays showed thatsignificant cell death occurred during this time accompaniedby an increased expression of caspase–3. Thrombin (10nM) stimulated cell growth with an accompanying increase inthe level of ERK and Akt phosphorylation. These increases wereinhibited in the cells where knock down had occurred, but notin cells exposed to scrambled control siRNA.

Conclusions: :
This study establishes a central role for the Sig–1Rin lens cell death and survival through modulating the activityof the ERK, Akt and caspase pathways.